Abstract HIV infection establishes a cellular reservoir that persists even after active and effective antiretroviral therapy is introduced. The best research approach to pursue HIV cure remains unsettled. The low levels of HIV-1 viremia observed in clinical studies of HDACi raise questions as to precisely what fraction of latently infected cells are induced to reactivate. Virion production may or may not be necessary for cure but, at a minimum, the synthesis of virus proteins is an absolute requirement for any strategy that leverages humoral or cell-mediated immunity to eradicate HIV. Here we note several critical knowledge gaps: the types, amount, and kinetics of LRA-induced HIV-1 translation are unknown. Flow cytometry, traditionally used to quantify cell protein expression, is insufficiently sensitive to detect HIV-1 protein production after latency reversal. Ribosome profiling is a novel technique that uses deep sequencing to characterize the nuclease footprints of ribosomes on mRNA transcripts in vivo. Protein synthesis can be monitored genome-wide and, when combined with mRNA-seq, provides a quantitative measure of translation efficiency. Here we propose to leverage an innovative technology to address important knowledge gaps in the HIV-1 eradication field and comprehensively assess LRA-induced HIV-1 translation across the genome. We hypothesize that ribosome profiling will identify lower translation efficiencies of the non-immune activating LRA, when compared to LRA that activate T cells. We will use lab-adapted latency cell lines to validate ribosome profiling and then apply the technique to LRA-treated resting CD4+ T cells isolated from ART-treated, virologically suppressed HIV-infected participants. We will first use the latently infected cell lines ACH-2 and J89 to quantify total (host + HIV-1) cellular mRNA by mRNA-seq and translation by ribosome footprint profiling and determine the effects of HIV-1 reactivation with PMA/ionomycin or the HDACi romidepsin. We will develop a series of computational tools to align, analyze, and interpret the combined RNA-seq and ribosome profiling datasets, in the context of host and HIV-1 products. Quality control measurements will be assessed and relative translation efficiencies, defined generally as translation divided by transcription, will be calculated. We will then define the genome-wide HIV-1 translation efficiencies in LRA-treated CD4+ ex vivo. Resting CD4+ T cells (rCD4) will be obtained by leukapheresis from six participants enrolled in the HIV Eradication and Latency (HEAL) cohort at BWH. Traditional measures of HIV persistence in blood will be quantified. rCD4 will be incubated in the presence or absence of an LRA panel. Ribosome profiling and mRNAseq will be performed. We will explore the relationships between translation efficiencies and LRA and relate these to measurements of total and infectious HIV-1 reservoir size.